DNA damage induces a variety of responses including, oxidative stress, apoptosis and premature senescence. These responses have been implicated both in aging and cancer. During the previous funding period, we observed evidence for significant roles of the nucleotide excision repair (NER) protein DDB2, which is mutated in xeroderma pigmentosum and under-expressed in squamous cell carcinoma, in DNA damage response. First, DDB2 plays an essential role in apoptosis induced by DNA damaging agents. In the absence of DDB2, DNA damaging agents cause cells to accumulate p21 at a very high level, leading to an inhibition of apoptosis. Deletion or depletion of p21 restores apoptosis in DDB2-deficient cells, indicating that the DDB2-mediated regulation of p21 is critical for apoptosis following DNA damage. Because of p21 accumulation, we investigated premature senescence. Surprisingly, the DDB2-/- MEFs escape senescence at a high frequency. Moreover, DDB2-/- MEFs or DDB2-depleted human cells do not exhibit oxidative stress in that they do not accumulate significant level of reactive oxygen species (ROS) following DNA damage. The lack of oxidative stress results from high-level expression of the antioxidant genes MnSOD and catalase. We observed evidence that DDB2 transcriptionally inhibits expression of MnSOD and catalase. Thus, in addition to characterizing its role in NER, we identified two new functions of DDB2 (proteolysis of p21 and transcriptional inhibition of the antioxidant genes) that are critical in apoptosis, oxidative stress and premature senescence following DNA damage. DDB2 associates with the E3 ubiquitin ligase Cul4-DDB1, which has been implicated also in gene regulation. In the current proposal, we will determine the mechanism that activates the DDB2-containing Cul4-DDB1 complex to target p21 for ubiquitination following DNA damage. Also, we will test the hypothesis that the DDB2- containing Cul4-DDB1 complex plays a central role in mediating oxidative stress following DNA damage by repressing expression of the antioxidant genes. The hypotheses will be tested through the following specific aims: 1. How does DDB2 induce proteolysis of p21 after DNA damage? 2. What is the mechanism by which DDB2 regulates the antioxidant genes? 3. Is DDB2 a mediator of oxidative stress and premature senescence following DNA damage? 4. Does the impaired premature senescence contribute to UVB-induced skin carcinogenesis in the DDB2-deficient mice? PUBLIC HEALTH RELEVANCE: Health Relevance: DDB2 was identified as the product of the XP-E gene, which is mutated in xeroderma pigmentosum. It is under-expressed in many cancers, including squamous cell carcinoma (Skin and Head & Neck). We found that, in addition to its role in DNA repair, DDB2 has other functions that are significant in cancer and aging. In this application, we plan to investigate the cellular pathways that are modulated by DDB2 that protect from DNA damage-induced mutations, and prevent development of squamous cell carcinoma.